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1.
Cells ; 9(9)2020 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-32899484

RESUMO

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.


Assuntos
Biopolímeros/química , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/metabolismo , Transfecção/métodos , Animais , Linhagem Celular , Ácidos Graxos Monoinsaturados/química , Feminino , Heparina/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica , Tamanho da Partícula , Compostos de Amônio Quaternário/química , RNA Mensageiro/química
2.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
3.
PLoS One ; 15(7): e0214497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639963

RESUMO

The Bashbay sheep (Ovis aries), an indigenous breed of Xinjiang, China, has many excellent characteristics. It is resistant to Mycoplasma ovipneumoniae infection, the causative agent of mycoplasma ovipneumonia, a chronic respiratory disease that is harmful to the sheep industry. To date, knowledge regarding the mechanisms responsible for M. ovipneumoniae pathogenesis in scant. Herein, we report the results of transcriptome profiling of lung tissues from Bashbay sheep experimentally infected with an M. ovipneumoniae strain at 4 and 14 days post-infection, in comparison to mock-infected animals (0 d). Transcriptome profiling was performed by deep RNA sequencing, using the Illumina platform. The analysis of differentially expressed genes was performed to determine concomitant gene-specific temporal patterns of mRNA expression in the lungs after M. ovipneumoniae infection. We found 1048 differentially expressed genes (575 up-regulated, 473 down-regulated) when comparing transcriptomic data at 4 and 0 days post-infection, and 2823 (1362 up-regulated, 1461 down-regulated) when comparing 14 versus 0 days post-infection. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed genes at 4 and 14 versus 0 days post-infection were enriched in 245 and 287 pathways, respectively, and the Toll-like receptor (TLR) signaling pathway was considered most closely related to MO infection (p < 0.01). Two pathways (LAMP-TLR2/TLR6-MyD88-MKK6-AP1-IL1B and LAMP-TLR8MyD88-IRF5-RANTES) were identified based on the TLR signaling pathway from differentially expressed genes related M. ovipneumoniae infection. Gene Ontology analysis showed that differentially expressed genes in different groups were enriched for 1580 and 4561 terms, where those most closely related to M. ovipneumoniae infection are positive regulators of inflammatory responses (p < 0.01). These results could aid in understanding how M. ovipneumoniae infection progresses in the lungs and may provide useful information regarding key regulatory pathways.


Assuntos
Pulmão/metabolismo , Pneumonia por Mycoplasma/patologia , Análise de Sequência de RNA/métodos , Doenças dos Ovinos/patologia , Transcriptoma , Animais , Regulação para Baixo , Mycoplasma ovipneumoniae/isolamento & purificação , Mycoplasma ovipneumoniae/patogenicidade , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/veterinária , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Regulação para Cima
4.
Nat Commun ; 11(1): 3392, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636376

RESUMO

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Quadruplex G , Estruturas R-Loop , Transcrição Genética , Proteínas Virais/genética , DNA/análise , DNA/química , RNA Polimerases Dirigidas por DNA/química , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , RNA/química , RNA Mensageiro/química , Sítio de Iniciação de Transcrição , Proteínas Virais/química
5.
Nucleic Acids Res ; 48(15): 8645-8662, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32614436

RESUMO

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.


Assuntos
Polinucleotídeo Adenililtransferase/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Poliadenilação/genética , Proteínas de Protozoários/química , Edição de RNA/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Protozoário/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
6.
Nucleic Acids Res ; 48(14): 7700-7711, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652016

RESUMO

Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current 'RNA-sequencing' technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00-6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.


Assuntos
Arabidopsis/genética , Sequenciamento por Nanoporos/métodos , RNA de Plantas/química , RNA de Plantas/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Citosina/metabolismo , Metilação , Isoformas de RNA/química , Isoformas de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA-Seq
7.
Proc Natl Acad Sci U S A ; 117(32): 19237-19244, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723815

RESUMO

The 5' messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3' end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3' to 5' degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.


Assuntos
Endorribonucleases/metabolismo , RNA Mensageiro/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo
8.
Nat Commun ; 11(1): 2823, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499480

RESUMO

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Neisseria meningitidis/genética , Conformação de Ácido Nucleico , Estresse Oxidativo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
9.
Nucleic Acids Res ; 48(12): 6491-6502, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484544

RESUMO

Multifunctional proteins often perform their different functions when localized in different subcellular compartments. However, the mechanisms leading to their localization are largely unknown. Recently, 3'UTRs were found to regulate the cellular localization of newly synthesized proteins through the formation of 3'UTR-protein complexes. Here, we investigate the formation of 3'UTR-protein complexes involving multifunctional proteins by exploiting large-scale protein-protein and protein-RNA interaction networks. Focusing on 238 human 'extreme multifunctional' (EMF) proteins, we predicted 1411 3'UTR-protein complexes involving 54% of those proteins and evaluated their role in regulating protein cellular localization and multifunctionality. We find that EMF proteins lacking localization addressing signals, yet present at both the nucleus and cell surface, often form 3'UTR-protein complexes, and that the formation of these complexes could provide EMF proteins with the diversity of interaction partners necessary to their multifunctionality. Our findings are reinforced by archetypal moonlighting proteins predicted to form 3'UTR-protein complexes. Finally, the formation of 3'UTR-protein complexes that involves up to 17% of the proteins in the human protein-protein interaction network, may be a common and yet underestimated protein trafficking mechanism, particularly suited to regulate the localization of multifunctional proteins.


Assuntos
Regiões 3' não Traduzidas , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , Proteínas de Membrana/química , Ligação Proteica , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas , Transporte Proteico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química
10.
Nucleic Acids Res ; 48(14): e81, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32504488

RESUMO

RNA secondary structure around translation initiation sites strongly affects the abundance of expressed proteins in Escherichia coli. However, detailed secondary structural features governing protein abundance remain elusive. Recent advances in high-throughput DNA synthesis and experimental systems enable us to obtain large amounts of data. Here, we evaluated six types of structural features using two large-scale datasets. We found that accessibility, which is the probability that a given region around the start codon has no base-paired nucleotides, showed the highest correlation with protein abundance in both datasets. Accessibility showed a significantly higher correlation (Spearman's ρ = 0.709) than the widely used minimum free energy (0.554) in one of the datasets. Interestingly, accessibility showed the highest correlation only when it was calculated by a log-linear model, indicating that the RNA structural model and how to utilize it are important. Furthermore, by combining the accessibility and activity of the Shine-Dalgarno sequence, we devised a method for predicting protein abundance more accurately than existing methods. We inferred that the log-linear model has a broader probabilistic distribution than the widely used Turner energy model, which contributed to more accurate quantification of ribosome accessibility to translation initiation sites.


Assuntos
Escherichia coli/genética , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas , Algoritmos , Códon de Iniciação/metabolismo , Conjuntos de Dados como Assunto , Escherichia coli/metabolismo , Previsões , Modelos Lineares , Aprendizado de Máquina , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico , Relação Estrutura-Atividade
11.
PLoS Comput Biol ; 16(5): e1007852, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379750

RESUMO

Single nucleotide polymorphisms are widely associated with disease, but the ways in which they cause altered phenotypes are often unclear, especially when they appear in non-coding regions. One way in which non-coding polymorphisms could cause disease is by affecting crucial RNA-protein interactions. While it is clear that changing a protein binding motif will alter protein binding, it has been shown that single nucleotide polymorphisms can affect RNA secondary structure, and here we show that single nucleotide polymorphisms can affect RNA-protein interactions from outside binding motifs through altered RNA secondary structure. By using a modified version of the Vienna Package and PAR-CLIP data for HuR (ELAVL1) in humans we characterize the genome-wide effect of single nucleotide polymorphisms on HuR binding and show that they can have a many-fold effect on the affinity of HuR binding to RNA transcripts from tens of bases away. We also find some evidence that the effect of single nucleotide polymorphisms on protein binding might be under selection, with the non-reference alleles tending to make it harder for a protein to bind.


Assuntos
Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Genoma Humano , Humanos , Ligação Proteica , RNA Mensageiro/química
12.
PLoS Genet ; 16(5): e1008787, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392243

RESUMO

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Regiões 5' não Traduzidas , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Células HeLa , Recombinação Homóloga , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química
13.
Nat Commun ; 11(1): 2118, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355211

RESUMO

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cell-free RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The single-cell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine.


Assuntos
RNA Mensageiro/química , Análise de Sequência de RNA , Análise de Célula Única/métodos , Células-Tronco/citologia , Células 3T3 , Animais , Antineoplásicos/farmacologia , Diferenciação Celular , Sistema Livre de Células , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células K562 , Camundongos , Medicina de Precisão , Reprodutibilidade dos Testes
14.
Nucleic Acids Res ; 48(10): 5684-5694, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356894

RESUMO

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate ß-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Proliferação de Células , Desmetilação , Células HEK293 , Células HeLa , Humanos , Oncogenes , Prevotella/enzimologia , Engenharia de Proteínas , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/metabolismo
15.
Nucleic Acids Res ; 48(12): 6943-6953, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463452

RESUMO

ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.


Assuntos
Proteínas Nucleares/genética , RNA Helicases/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HEK293 , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas Nucleares/química , Domínios Proteicos/genética , RNA Helicases/química , RNA Mensageiro/química , RNA Nuclear/química , RNA Nuclear/genética
16.
Nucleic Acids Res ; 48(11): 6136-6148, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374864

RESUMO

In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3'-FADP or CoA and fission yeast Rai1 with 3'-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.


Assuntos
Coenzima A/metabolismo , Exorribonucleases/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Kluyveromyces/enzimologia , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Animais , Flavina-Adenina Dinucleotídeo/análise , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , NAD/metabolismo , Capuzes de RNA/análise , Especificidade por Substrato , Transcrição Genética
17.
Nucleic Acids Res ; 48(W1): W239-W243, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32421834

RESUMO

Recent evidences suggest that the localization of mRNAs near the subcellular compartment of the translated proteins is a more robust cellular tool, which optimizes protein expression, post-transcriptionally. Retention of mRNA in the nucleus can regulate the amount of protein translated from each mRNA, thus allowing a tight temporal regulation of translation or buffering of protein levels from bursty transcription. Besides, mRNA localization performs a variety of additional roles like long-distance signaling, facilitating assembly of protein complexes and coordination of developmental processes. Here, we describe a novel machine-learning based tool, mRNALoc, to predict five sub-cellular locations of eukaryotic mRNAs using cDNA/mRNA sequences. During five fold cross-validations, the maximum overall accuracy was 65.19, 75.36, 67.10, 99.70 and 73.59% for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. Assessment on independent datasets revealed the prediction accuracies of 58.10, 69.23, 64.55, 96.88 and 69.35% for extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The corresponding values of AUC were 0.76, 0.75, 0.70, 0.98 and 0.74 for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The mRNALoc standalone software and web-server are freely available for academic use under GNU GPL at http://proteininformatics.org/mkumar/mrnaloc.


Assuntos
RNA Mensageiro/análise , Software , Máquina de Vetores de Suporte , Núcleo Celular/química , Simulação por Computador , Citoplasma/química , Retículo Endoplasmático/química , Mitocôndrias/química , RNA Mensageiro/química , Análise de Sequência de RNA
18.
Gene ; 745: 144640, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32247037

RESUMO

Codon usage bias is an important genomic phenomenon, where highly expressed genes use optimal codons for smoother translation with high yield, facilitated by the cognate tRNAs. Here, we presented the tRNA co-adaptation index (co-AI) by correlating tRNA gene copy number and codon composition in Saccharomyces cerevisiae. We observed that this co-AI is positively correlated with protein abundance and translation rate. Considering nucleotide substitutions, co-AI influences synonymous substitutions more than gene expression and protein abundance, the most important determinants of evolutionary rate. Co-AI correlates positively with mRNA secondary structure stability and mRNA half-life, which may lead to protein accumulation under high co-AI. However, the highly expressed proteins encoded by high co-AI genes are assisted by molecular chaperones to attain their proper functional conformation and prevent accumulation.


Assuntos
Dosagem de Genes , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Mutação Silenciosa , Uso do Códon , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
19.
New Microbiol ; 43(2): 58-63, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32310297

RESUMO

Up to now, the UL16-17 region of human cytomegalovirus (HCMV) has not been well characterized at the level of mRNA and protein, especially for the Han strain, the first clinical HCMV strain in China. In previous studies, three transcripts were detected from the UL16-17 region by northern blot analysis for Merlin strain. Transcriptions of UL16 and UL17 were also studied by 5' rapid amplification of cDNA ends (5'RACE) and deep sequencing for AD169 and Towne strains, respectively. However, details of 3' end of UL16 and UL17 transcripts have never been confirmed by 3'RACE. The expressing phage of the UL16-17 region needs further research by northern blot, too. In the present study, cDNA library screening, northern blot and RACE were used to identify the transcription characteristics of the UL16-17 region. Mainly, 3 clusters of transcripts with the same 3' end were found to be expressed from the UL16-17 region in both Han and AD169 strains. The lengths of the core transcripts among the 3 clusters were 1,254nt, 718nt and 468nt, respectively. The corresponding 5' ends are at nt23119, nt23655, nt23905 in the HCMV Han genome. The consistent 3' end is located at nt24372 in the Han genome. The 1,254nt and 468nt transcripts are transcribed in early and late phases, and the 718nt transcript is transcribed only in the late phase.


Assuntos
Citomegalovirus , Proteínas Virais , China , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Biblioteca Gênica , Humanos , RNA Mensageiro/química , Proteínas Virais/química , Proteínas Virais/genética
20.
Nucleic Acids Res ; 48(10): e59, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32286626

RESUMO

Methods to measure heterogeneity among cells are rapidly transforming our understanding of biology but are currently limited to molecular abundance measurements. We developed an approach to simultaneously measure biochemical activities and mRNA abundance in single cells to understand the heterogeneity of DNA repair activities across thousands of human lymphocytes, identifying known and novel cell-type-specific DNA repair phenotypes.


Assuntos
Reparo do DNA , Expressão Gênica , Análise de Célula Única/métodos , Linhagem Celular , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfócitos/metabolismo , Fenótipo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA-Seq , Análise de Sequência de DNA
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